Brief periods of stress have been shown to initiate protective signaling mechanisms, that reduce injury during subsequent more severe or sustained injury. The goals of this project are to elucidate signaling mechanisms involved in this protection. Hypoxic via a transcription factor, hypoxic inducible factor-1-alpha (HIF-1-alpha), has been known to upregulate a number of hypoxic sensitive genes. The mechanism by oxygen regulates levels of HIF-1-alpha has been shown to be via hydroxylation of a proline in HIF-1-alpha that targets it for degradation. During hypoxia, the cessation of hydroxylation of this proline results in less degradation and thus increases HIF-1-alpha protein levels. We examined the consequences of activating the PHD oxygen-sensing pathway in cultured neonatal myocytes using ethyl-3,4 dihydroxybenzoate (EDHB) and dimethyloxalylglycine (DMOG), inhibitors that, similar to hypoxia, inhibit O2-dependent PHD enzymes. We found that these inhibitors lead to upregulation of glucose transport, nitric oxide synthase and heme oxygenase. We also made the novel observation that inhibition of these prolyl hydroxylase leads to protection against subsequent cell death due to addition of metabolic toxins. We have also investigated protective signaling mechanisms that do not require new protein synthesis, but instead confer protection via post-translation signaling mechanisms. Brief intermittent stress (termed preconditioning) can lead to release of cytokines and hormones such as adenosine, opioids, bradykinin, and other peptides which through activation of G protein coupled receptors leads to protection. We found that inhibition of PI3K, with wortmannin blocked the protection afforded by preconditioning. To examine whether PI3K was activated by Gg, we tested whether sequestering Gg blocks preconditioning. We find that preconditioning requires signaling through Gg, as we find that hearts from mice with cardiac specific overexpression of a peptide that binds Gg (BARKct) are not protected after preconditioning. We have begun to elucidate the signaling mechanism by which Gg initiates protection. We found that inhibition of Gg signaling does not block preconditioning induced activation of the PI3K pathway. The PC-induced phosphorylation of p70 S6K or PKB, kinases downstream of PI3K were not blocked in TG bARKct hearts. We therefore investigated other targets of PI3K. Gbg and PI3K are both involved in receptor recycling and potential endosomal signaling. PI3K and the lipid products phosphoinositides play a critical role in recruitment of b-arrestin-GPCRs complexes to endosomes. A recent study has demonstrated that PI3K regulates GPCR trafficking by promoting the recruitment of AP-2 to the receptor/b-arrestin complex, thus potentially enhancing recycling of internalized receptors to the cell surface. b-arrestin has recently been shown to be a scaffolding protein that brings other signaling proteins in contact with GPCRs in the endosome during receptor recycling. There is increasing evidence that in addition to leading to receptor desensitization, the bARK (GRK)/b-arrestin interaction can lead to activation of signals such as the MAPK pathway (ERK and JNK). Consistent with this hypothesis we have made the novel observation that inhibitors of endocytosis also block protection afforded by preconditioning and we are pursuing the hypothesis that Gg blocks preconditioning by inhibiting endocytosis and resulting cell signals. We have also investigated the role of inhibition of apoptosis in cardioprotection. The anti-apoptotic protein Bcl-2 is targeted to the mitochondria, but it is uncertain whether Bcl-2 affects only myocyte survival following ischemia, or also metabolic functions of mitochondria during ischemia. To study the effect of Bcl-2 on energetics and intracellular pH (pHi), 31P-NMR spectroscopy was used with isolated perfused hearts from mice overexpressing human Bcl-2 (Bcl-2) and from their wild-type littermates (WT). Hearts were subjected to 24 minutes of global zero-flow ischemia at 37oC followed by reperfusion. During ischemia, the fall in pHi and the initial rate of decline in ATP were significantly reduced in Bcl-2 hearts compared with WT hearts (p0.05) and in Bcl-2 hearts Oligo had no additional effect on pHi during ischemia. Addition of Oligo to WT hearts slowed the rate of decline in ATP during ischemia to a level similar to that observed in Bcl-2 hearts, but addition of Oligo had no significant effect on the rate of decline in ATP in Bcl-2 hearts during ischemia. These observations are consistent with Bcl-2 induced closure of the voltage-dependent anion channel (VDAC), an outer mitochondrial membrane channel responsible for providing ATP to F1F0-ATPase. Immunoprecipitation showed greater interaction between Bcl-2 and VDAC in Bcl-2 hearts compared to WT hearts. Thus, the modulation of VDAC by Bcl-2 during ischemia may be an important mechanism for Bcl-2-induced cardioprotection. We have also examined failing human hearts with the goal of identifying gene-environment interactions that may predispose the hearts to congestive heart failure. We used DNA microarray profiling of human heart tissue to investigate changes in the expression of genes involved in apoptosis that occur in human idiopathic dilated cardiomyopathic hearts that had progressed to heart failure. We found a pro-apoptotic shift in the tumor necrosis factor-alpha pathway which is involved in myocyte loss and decompensated heart failure. Specifically we found decreased expression of TNF-alpha and NF-kappaB induced anti-apoptotic genes such as growth arrest and DNA damage inducible (GADD) 45, Flice inhibitory protein (FLIP), and TNF-induced protein 3. Consistent with a role for apoptosis in heart failure, we also observed a decrease in phosphorylation of BAD at Ser-112. This study identified several pathways that are altered in human heart failure and provides new targets for therapy.